LOCUS       sequence-224406-        4342 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for PCR amplification of mWasabi DNA template for in vitro 
            transcription.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4342)
  AUTHORS   McGrath PS, Diette N, Kogut I, Bilousova G
  TITLE     RNA-based Reprogramming of Human Primary Fibroblasts into Induced 
            Pluripotent Stem Cells.
  JOURNAL   J Vis Exp. 2018 Nov 26;(141). doi: 10.3791/58687.
  PUBMED    30531717
REFERENCE   2  (bases 1 to 4342)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Vis 
            Exp."; date: "2018-11-26"; volume: "(141). doi"; pages: " 
            10.3791/58687"
COMMENT     Sequence Label: pCR-Blunt_mWasabi
FEATURES             Location/Qualifiers
     source          1..4342
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1078..1096
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1136..1145
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1142..1852
                     /codon_start=1
                     /product="green variant of teal fluorescent protein mTFP1 
                     (Ai et al., 2008)"
                     /label=mWasabi
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFSYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKETTGWDASTERMYVRDGVLK
                     GDVKMKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYEIA
                     VARNSTDGMDELYK"
     promoter        complement(2001..2019)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2026..2043)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2026..2042)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2035..2057)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2180..2482
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2361..2380
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2831..3625
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2831..3625
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2885..2904)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3495..3514
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3825..3834
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3831..4205
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat ttaggtgacg
     1021 cgttagaata ctcaagctat gcatcaagct tggtaccttg gaccctcgta cagaagctaa
     1081 tacgactcac tatagggaaa taagagagaa aagaagagta agaagaaata taagagccac
     1141 catggtgagc aagggcgagg agaccacaat gggcgtaatc aagcccgaca tgaagatcaa
     1201 gctgaagatg gagggcaacg tgaatggcca cgccttcgtg atcgagggcg agggcgaggg
     1261 caagccctac gacggcacca acaccatcaa cctggaggtg aaggagggag cccccctgcc
     1321 cttctcctac gacattctga ccaccgcgtt cagttacggc aacagggcct tcaccaagta
     1381 ccccgacgac atccccaact acttcaagca gtccttcccc gagggctact cttgggagcg
     1441 caccatgacc ttcgaggaca agggcatcgt gaaggtgaag tccgacatct ccatggagga
     1501 ggactccttc atctacgaga tacacctcaa gggcgagaac ttccccccca acggccccgt
     1561 gatgcagaag gagaccaccg gctgggacgc ctccaccgag aggatgtacg tgcgcgacgg
     1621 cgtgctgaag ggcgacgtca agatgaagct gctgctggag ggcggcggcc accaccgcgt
     1681 tgacttcaag accatctaca gggccaagaa ggcggtgaag ctgcccgact atcactttgt
     1741 ggaccaccgc atcgagatcc tgaaccacga caaggactac aacaaggtga ccgtttacga
     1801 gatcgccgtg gcccgcaact ccaccgacgg catggacgag ctgtacaagt aagctgcctt
     1861 ctgcggggct tgccttctgg ccatgccctt cttctctccc ttgcacctgt acctcttggt
     1921 ctttgaataa agcctggaat tctgcagata tccatcacac tggcggccgc tcgagcatgc
     1981 atctagaggg cccaattcgc cctatagtga gtcgtattac aattcactgg ccgtcgtttt
     2041 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     2101 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     2161 gcgcagccta tacgtacggc agtttaaggt ttacacctat aaaagagaga gccgttatcg
     2221 tctgtttgtg gatgtacaga gtgatattat tgacacgccg gggcgacgga tggtgatccc
     2281 cctggccagt gcacgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca
     2341 tatcggggat gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtctccgt
     2401 tatcggggaa gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa
     2461 cctgatgttc tggggaatat aaatgtcagg catgagatta tcaaaaagga tcttcaccta
     2521 gatccttttc acgtagaaag ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag
     2581 ctactgggct atctggacaa gggaaaacgc aagcgcaaag agaaagcagg tagcttgcag
     2641 tgggcttaca tggcgatagc tagactgggc ggttttatgg acagcaagcg aaccggaatt
     2701 gccagctggg gcgccctctg gtaaggttgg gaagccctgc aaagtaaact ggatggcttt
     2761 cttgccgcca aggatctgat ggcgcagggg atcaagctct gatcaagaga caggatgagg
     2821 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     2881 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     2941 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     3001 gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     3061 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     3121 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     3181 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     3241 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     3301 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag
     3361 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     3421 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     3481 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     3541 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     3601 tcgccttctt gacgagttct tctgaattat taacgcttac aatttcctga tgcggtattt
     3661 tctccttacg catctgtgcg gtatttcaca ccgcatcagg tggcactttt cggggaaatg
     3721 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3781 gacaataacc ctgataaatg cttcaataat agcacgtgag gagggccacc atggccaagt
     3841 tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga
     3901 ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg
     3961 acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg
     4021 cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca
     4081 cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc
     4141 gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg
     4201 actgacacgt gctaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     4261 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4321 tagaaaagat caaaggatct tc
//
